Use of a polypeptide for effecting immune signalling and/or affecting intestinal barrier function and/or modulating metabolic status

ABSTRACT

It has been found that an extracellular polypeptide derived from Akkermansia municiphila is capable of modulating and/or promoting gut mucosal immune system function and/or maintaining and/or restoring metabolic status and/or increasing the physical integrity of the gut mucosal barrier in a mammal. The polypeptide or host cells comprising such polypeptide may be employed to prevent and/or treat a variety of conditions that benefit from an increased physical integrity of the gut mucosal barrier and/or an improved gut mucosal immune system function and metabolic status.

FIELD OF THE INVENTION

The invention relates to the fields of gut mucosal immune system, gutmucosal barrier, pharmaceutical, food or feed compositions comprisingpolypeptides and/or host cells, which are capable of modulating and/orpromoting gut mucosal immune system function and/or maintaining and/orrestoring and/or increasing the physical integrity of the gut mucosalbarrier, and/or of maintaining, restoring or improving glucose and/orcholesterol and/or triglyceride homeostasis in a mammal (e.g. human).More specifically, the present invention provides compositionscomprising Amuc-1100 polypeptide, or variants thereof. It has been foundthat Amuc-1100 is capable of interacting with the toll-like receptor 2(TLR2) and/or modulating TLR2 and/or the NFk-B-dependent signallingpathway, and/or promoting cytokine release (e.g. IL-6, IL-8, and IL-10)from immune cells located in the vicinity of the mucosal gut barrier ofa mammal (e.g. human), and/or is capable of maintaining, restoring orincreasing the physical integrity of the gut mucosal barrier and/or ofmaintaining, restoring or improving glucose and/or cholesterol and/ortriglyceride homeostasis in a mammal and/or is capable of improving themetabolic or immune status of a mammal. The Amuc-1100 polypeptide may beused to prevent or treat a variety of diseases or conditions as setforth herein.

BACKGROUND OF THE INVENTION

Increased permeability or hyperpermeability of the gut mucosal barrieris thought to play a role in several disorders and conditions such asbowel related diseases, autoimmune diseases, allergies, cancers, type 2diabetes, obesity, depression, anxiety, and many others. For thisreason, there has been an increased interest in understanding the roleof the gut mucosal barrier dysfunction in the pathogenesis of manyconditions targeting the gastrointestinal tract (GI) in mammals.

Under normal conditions, the gut mucosal barrier acts as a selectivebarrier permitting the absorption of nutrients, electrolytes and waterand preventing the exposure to detrimental macromolecules,micro-organisms, dietary and microbial antigens (e.g. food allergens).The gut mucosal barrier is essentially composed of a layer of mucus andan underlying layer epithelial cells (referred to herein as ‘gutepithelial cells’). The gut epithelial cells are tightly linked to eachother by so-called ‘tight junctions’, which are basically ‘physicaljoints’ between the membranes of two gut epithelial cells. Maintenanceof the gut mucosal barrier, particularly maintenance of the physicalintegrity of the gut epithelial cell layer (i.e. keeping the junctionsbetween cell tight), is crucial for protection of the host against themigration of pathogenic micro-organisms, antigens, and other undesirableagents from the intestine to the blood stream.

The gut mucosal barrier is also heavily colonized by approximately10¹²-10¹⁴ commensal microorganisms, mainly anaerobic or microaerophilicbacteria, most of which live in symbiosis with their host. Thesebacteria are beneficial to their host in many ways. They provideprotection against pathogenic bacteria and serve a nutritional role intheir host by synthesizing vitamin K and some of the components of thevitamin B complex. Further, the gut mucosal barrier has evolved acomplex ‘gut mucosal immune system’ for distinguishing between commensal(i.e. beneficial bacteria) and pathogenic bacteria and other detrimentalagents. The gut mucosal immune system is an integral part of the gutmucosal barrier, and comprises lymphoid tissues and specialized immunecells (i.e. lymphocytes and plasma cells), which are scattered widelythroughout the gut mucosal barrier. One of the microorganisms thatnaturally colonizes the mucosa of healthy subjects is themucin-degrading Akkermansia muciniphila, which has been shown toincrease the intestinal barrier function (Everard et al., PNAS 110(2013) 9066-71; Reunanen et al., Appl Environ Microbiol Mar. 20, 2015),and thereby impact diseases associated with impaired gut barrierfunction.

Under certain circumstances, the gut mucosal barrier may be vulnerableto a wide variety of infectious organisms or agents, which are normallynot able to cross the mucosal gut barrier but nevertheless manage tocross it (e.g. through gaps resulting from loose tight junctions betweengut epithelia cells). Organisms or other agents that cross the gutmucosal barrier may cause diseases or other undesirable conditions (e.g.allergies) in the host. Examples of such diseases include obesity,metabolic syndrome, insulin-deficiency or insulin-resistance relateddisorders, type 2 diabetes, type 1 diabetes, inflammatory bowel disease(IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormallipid metabolism, atherosclerosis, hypertension, cardiac pathology,stroke, non-alcoholic fatty liver disease, alcoholic fatty liverdisease, hyperglycemia, hepatic steatosis, dyslipidaemias, dysfunctionof the immune system associated with obesity (weight gain), allergy,asthma, autism, parkinson's disease, multiple sclerosis,neurodegenerative diseases, depression, other diseases related tocompromised barrier function, wound healing, behavioural disorders,alcohol dependence, cardiovascular diseases, high cholesterol, elevatedtriglycerides, atherosclerosis, sleep apnoea, osteoarthritis,gallbladder disease, and cancer.

Conversely, diseases such as those mentioned above as well as otherconditions such as food allergies, immaturity of the gut, e.g., due to ababy being born prematurely, exposure to radiation, chemotherapy and/ortoxins, autoimmune disorders, malnutrition, sepsis, and the like, mayalter the physical integrity of the gut mucosal barrier (i.e. causeloosening of the tight junctions between the gut epithelial cells),which in turn may allow undesirable micro-organism or other agents tocross the host gut mucosal barrier.

Several vaccines and/or antibodies targeted against such micro-organismsor agents have been developed over the years. However, the success ofsuch approach is mitigated as several micro-organisms or agents cannotbe effectively targeted or eradicated with vaccines or antibodies.

Other approaches, which aim at preventing detrimental micro-organismsand other agents to cross the host's gut mucosal barrier in the firstplace and/or aim at preventing hyperpermeability of the gut mucosalbarrier, have also been explored. For instance, compositions comprisingglutamic acid have been developed to prevent and/or treat conditionsassociated with hyperpermeability of the gut mucosal barrier (WO01/58283). Other substances including spermine and spermidine andprecursors thereof, have also been used for the same purpose (Dorhout etal (1997). British J. Nutrition, pages 639-654). Preparations comprisingarabinoxylan for promoting beneficial effects on the GI bacteria livingin the vicinity of the gut mucosal barrier, have also been developed forthe purpose of modulating the gut mucosal barrier (US2012/0230955).

It is an object of the present invention to provide agents and/orcompositions comprising such agents, which are suitable for maintainingand/or restoring and/or increasing the physical integrity of the gutmucosal barrier and/or preventing hyperpermeability of the gut mucosalbarrier in a mammal (e.g. human), and/or for maintaining and/orrestoring and/or improving glucose and/or cholesterol and/ortriglyceride homeostasis in a mammal, and preferably thereby prevent ortreat diseases or conditions that are associated with suboptimalpermeability of the gut mucosal barrier and/or glucose and/orcholesterol and/or triglyceride homeostasis imbalance in said mammal.Alternatively or additionally, it is an object of the present inventionto provide agents and/or compositions comprising such agents, which aresuitable for modulating and/or promoting the gut mucosal immune systemfunction in a mammal.

SUMMARY OF THE INVENTION

The present invention is concerned with a composition comprising apolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or anamino acid sequence comprising at least 50% sequence identity to theamino acid sequence of SEQ ID NO:1 over the entire length, saidpolypeptide being capable of effecting immune signaling and/or affectingintestinal barrier function and/or affecting glucose and/or cholesteroland/or triglyceride homeostasis, and a pharmaceutically or alimentarilyacceptable carrier.

The composition may be a nutritional composition or a pharmaceuticalcomposition.

The invention is also related to a genetically modified host cellwherein a nucleic acid molecule selected from the group of: a) a nucleicacid molecule comprising a nucleic acid sequence having at least 50%sequence identity with SEQ ID NO: 2 over the entire length; and b) anucleic acid molecule comprising a nucleic acid sequence that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or anamino acid sequence comprising at least 50% sequence identity to theamino acid sequence of SEQ ID NO:1 over the entire length, saidpolypeptide being capable of effecting immune signaling and/or affectingintestinal barrier function and/or affecting glucose and/or cholesteroland/or triglyceride homeostasis, is introduced into its genome.

Additionally, the invention is directed to a genetically modified hostcell, said host cell not being of the species Akkermansia muciniphilla,comprising a nucleic acid molecule selected from the group of: a) anucleic acid molecule comprising nucleic acid sequence having at least50% sequence identity with SEQ ID NO: 2 over the entire length; and b) anucleic acid molecule comprising a nucleic acid sequence that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or anamino acid sequence comprising at least 50% sequence identity to theamino acid sequence of SEQ ID NO:1 over the entire length, saidpolypeptide being capable of effecting immune signaling and/or affectingintestinal barrier function and/or affecting glucose and/or cholesteroland/or triglyceride homeostasis.

The invention further provides a genetically modified host cell, saidhost cell being of the species Akkermansia muciniphila, wherein anucleic acid molecule selected from the group of: a) a nucleic acidmolecule comprising a nucleic acid sequence having at least 50% sequenceidentity with SEQ ID NO: 2 over the entire length; and b) a nucleic acidmolecule comprising a nucleic acid sequence that encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or an amino acidsequence comprising at least 50% sequence identity to the amino acidsequence of SEQ ID NO:1 over the entire length, said polypeptide beingcapable of effecting immune signaling and/or affecting intestinalbarrier function and/or affecting glucose and/or cholesterol and/ortriglyceride homeostasis, is introduced into its genome.

The invention further pertains to a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or an amino acidsequence comprising at least 50% sequence identity to the amino acidsequence of SEQ ID NO:1 over the entire length, said polypeptide beingcapable of effecting immune signaling and/or affecting intestinalbarrier function and/or affecting glucose and/or cholesterol and/ortriglyceride homeostasis, comprising the steps of: a) culturing a hostcell according to any of claims 3-5 under conditions permittingproduction of said polypeptide; and b) optionally, isolating thepolypeptide produced in step (a).

The invention further provides for a polypeptide comprising the aminoacid sequence of SEQ ID NO: 1 or an amino acid sequence comprising atleast 50% sequence identity to the amino acid sequence of SEQ ID NO:1over the entire length, said polypeptide being capable of effectingimmune signaling and/or affecting intestinal barrier function and/oraffecting glucose and/or cholesterol and/or triglyceride homeostasis, ahost cell as taught herein, or a composition as taught herein, for useas a medicament, particularly for use in promoting gut mucosal immunesystem function, for maintaining, restoring and/or improving glucoseand/or cholesterol and/or triglyceride homeostasis, or for maintaining,restoring and/or increasing the physical integrity of the gut mucosalbarrier in a mammal.

Said polypeptide, composition or host cell may be for use in preventingand/or treating a disorder selected from the group consisting ofobesity, metabolic syndrome, insulin-deficiency or insulin-resistancerelated disorders, type 2 diabetes, type 1 diabetes, gestationaldiabetes, preeclampsia, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism,atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholicfatty liver disease, alcoholic fatty liver disease, hyperglycemia,hepatic steatosis, dyslipidaemias, dysfunction of the immune systemassociated with obesity (weight gain), allergy, asthma, autism,parkinson's disease, multiple sclerosis, neurodegenerative diseases,depression, other diseases related to compromised barrier function,wound healing, behavioural disorders, alcohol dependence, cardiovasculardiseases, high cholesterol, elevated triglycerides, atherosclerosis,sleep apnea, osteoarthritis, gallbladder disease, cancer, and conditionsaltering the physical integrity of the gut mucosal barrier such as foodallergies, immaturity of the gut, e.g., due to a baby being bornprematurely, exposure to radiation, chemotherapy and/or toxins,autoimmune disorders, malnutrition, sepsis, and the like, in a mammal.

Alternatively or additionally, said polypeptide, host cell orcomposition may be for use in promoting anti-inflammatory activity inthe gut of a mammal, and/or for use in promoting weight loss in amammal.

The invention also relates to a method for treating and/or preventing adisorder selected from the group of obesity, metabolic syndrome,insulin-deficiency or insulin-resistance related disorders, type 2diabetes, type 1 diabetes, gestational diabetes, preeclampsia,inflammatory bowel disease (IBD), irritable bowel syndrome (IBS),glucose intolerance, abnormal lipid metabolism, atherosclerosis,hypertension, cardiac pathology, stroke, non-alcoholic fatty liverdisease, alcoholic fatty liver disease, hyperglycemia, hepaticsteatosis, dyslipidaemias, dysfunction of the immune system associatedwith obesity (weight gain), allergy, asthma, autism, parkinson'sdisease, multiple sclerosis, neurodegenerative diseases, depression,other diseases related to compromised barrier function, wound healing,behavioural disorders, alcohol dependence, cardiovascular diseases, highcholesterol, elevated triglycerides, atherosclerosis, sleep apnoea,osteoarthritis, gallbladder disease, cancer, and conditions altering thephysical integrity of the gut mucosal barrier such as food allergies,immaturity of the gut, e.g., due to a baby being born prematurely,exposure to radiation, chemotherapy and/or toxins, autoimmune disorders,malnutrition, sepsis, and the like, in a mammal, for promoting weightloss in a mammal, for promoting anti-inflammatory activity in the gut ofa mammal, for promoting gut mucosal immune system function in a mammal,for maintaining, restoring and/or improving glucose and/or cholesteroland/or triglyceride homeostasis in a mammal, or for maintaining,restoring and/or increasing the physical integrity of the mucosal gutbarrier of a mammal, comprising the step of administering to a mammal inneed thereof, an effective amount of a polypeptide comprising the aminoacid sequence of SEQ ID NO: 1 or an amino acid sequence comprising atleast 50% sequence identity to the amino acid sequence of SEQ ID NO:1over the entire length, said polypeptide being capable of effectingimmune signaling and/or affecting intestinal barrier function and/oraffecting glucose and/or cholesterol and/or triglyceride homeostasis, ahost cell as taught herein, or a composition as taught herein.

The invention further pertains to a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or an amino acidsequence comprising at least 50% sequence identity to the amino acidsequence of SEQ ID NO:1 over the entire length, said polypeptide beingcapable of effecting immune signaling and/or affecting intestinalbarrier function and/or affecting glucose and/or cholesterol and/ortriglyceride homeostasis, comprising the steps of: a) culturing bacteriaof the species Akkermansia muciniphila in a suitable culture medium; andb) optionally, isolating the polypeptide produced in step (a).

General Definitions

In the context of the present invention, the term “polypeptide” isequivalent to the term “protein”. A polypeptide has a particular aminoacid sequence. A “variant” of the polypeptide of the present inventionpreferably has an amino acid sequence that has at least 50% sequenceidentity to the polypeptide of the present invention. A polypeptide ofthe invention is isolated when it is no longer in its naturalenvironment, i.e., when it is no longer present in the context offimbriae, and/or no longer present in the context of a cell, such as anAkkermansia muciniphila cell.

The term ‘sequence identity’ or ‘sequence similarity’ as used hereinrefer to a situation where an amino acid or a nucleic acid sequence hassequence identity or sequence similarity with another reference aminoacid or nucleic acid sequence. ‘Sequence identity’ or ‘sequencesimilarity’ can be determined by alignment of two polypeptides or twonucleotide sequences using global or local alignment algorithms.Sequences may then be referred to as “substantially identical” or“essentially similar” when they (when optimally aligned by for examplethe programs GAP or BESTFIT using default parameters) share at least acertain minimal percentage of sequence identity (as defined below). GAPuses the Needleman and Wunsch global alignment algorithm to align twosequences over their entire length, maximizing the number of matches andminimises the number of gaps. Generally, the GAP default parameters areused, with a gap creation penalty=50 (nucleotides)/8 (proteins) and gapextension penalty=3 (nucleotides)/2 (proteins). For nucleotides thedefault scoring matrix used is nwsgapdna and for proteins the defaultscoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89,915-919). Sequence alignments and scores for percentage sequenceidentity may be determined using computer programs, such as the GCGWisconsin Package, Version 10.3, available from Accelrys Inc., 9685Scranton Road, San Diego, Calif. 92121-3752 USA, or EmbossWin version2.10.0 (using the program “needle”). Alternatively percent similarity oridentity may be determined by searching against databases, usingalgorithms such as FASTA, BLAST, etc. Preferably, the sequence identityrefers to the sequence identity over the entire length of the sequence.

‘Transepithelial resistance’ (abbreviated as TER) is a measure of thepermeability of an epithelial cell layer in vitro. Increased epithelialpermeability has been linked to weakening of the tight junctions, andwith decrease of TER.

The term ‘chimeric gene’ as used herein refers to any non-naturallyoccurring gene, i.e., a gene which is not normally found in nature in aspecies, in particular a gene in which one or more parts of the nucleicacid sequence are not associated with each other in nature. For example,the promoter is not associated in nature with part or all of thetranscribed region or with another regulatory region. The term ‘chimericgene’ is understood to include expression constructs in which aheterologous promoter or transcription regulatory sequence is operablylinked to one or more coding sequences, and optionally a 3′-untranslatedregion (3′-UTR). Alternatively, a chimeric gene may comprise a promoter,coding sequence and optionally a 3′-UTR derived from the same species,but that do not naturally occur in this combination.

The term ‘genetically modified host cell’ as used herein refers to cellsthat have been genetically modified, e.g. by the introduction of anexogenous nucleic acid sequence (e.g. SEQ ID NO:2 as taught herein) orby specific alteration of an endogenous gene sequence. Such cells mayhave been genetically modified by the introduction of, e.g., one or moremutations, insertions and/or deletions in the endogenous gene and/orinsertion of a genetic construct (e.g. vector, or chimeric gene) in thegenome. Genetically modified host cells may refer to cells in isolationor in culture. Genetically modified cells may be ‘transduced cells’,wherein the cells have been infected with for instance a modified virus,e.g., a retrovirus may be used but other suitable viruses may also becontemplated such as lentiviruses. Non-viral methods may also be used,such as transfections. Genetically modified host cells may thus also be‘stably transfected cells’ or ‘transiently transfected cells’.Transfection refers to non-viral methods to transfer DNA (or RNA) tocells such that a gene is expressed. Transfection methods are widelyknown in the art, such as calcium-phosphate transfection, PEGtransfection, and liposomal or lipoplex transfection of nucleic acids,and the like. Such a transfection may be transient, but may also be astable transfection, wherein cells that have integrated the geneconstruct into their genome may be selected.

The term ‘effective amount’ as used herein refers to an amount necessaryto achieve an effect as taught herein. For instance, an effective amountof the polypeptide or genetically engineered host cell as taught herein,is an amount which is effectively useful for modulating and/or promotingthe gut mucosal immune system function and/or maintaining and/orrestoring and/or increasing the physical integrity of the gut mucosalbarrier (e.g., promoting formation of tighter junction between the gutepithelium cells), and/or for modulating and/or stimulating thetoll-like receptor signaling pathway (i.e. TLR2 pathway) in an immunecell and/or for increasing cytokine production (e.g. IL-6, IL-8, andIL-10) in an immune cell, and/or for preventing and/or treatingdisorders or conditions such as obesity, metabolic syndrome,insulin-deficiency or insulin-resistance related disorders, type 2diabetes, type 1 diabetes, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism,atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholicfatty liver disease, alcoholic fatty liver disease, hyperglycemia,hepatic steatosis, dyslipidaemias, dysfunction of the immune systemassociated with obesity (weight gain), allergy, asthma, autism,parkinson's disease, multiple sclerosis, neurodegenerative diseases,depression, other diseases related to compromised barrier function,wound healing, behavioural disorders, alcohol dependence, cardiovasculardiseases, high cholesterol, elevated triglycerides, atherosclerosis,sleep apnoea, osteoarthritis, gallbladder disease, cancer, andconditions altering the physical integrity of the gut mucosal barriersuch as food allergies, immaturity of the gut, e.g., due to a baby beingborn prematurely, exposure to radiation, chemotherapy and/or toxins,autoimmune disorders, malnutrition, sepsis, and the like.

The term ‘physiologically-acceptable carrier’ or ‘alimentarilyacceptable carrier’, ‘nutritionally acceptable carrier’ or‘pharmaceutically-acceptable carrier’ as used herein refers to aphysiologically-acceptable or alimentarily acceptable carrier ornutritionally-acceptable or pharmaceutically-acceptable carriermaterial, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in providing an administration formof the polypeptide or host cell of the invention. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the composition and not injurious to the subject, i.e. which aresuitable for consumption or nutritionally acceptable. The term ‘suitablefor consumption’ or ‘nutritionally acceptable’ refers to ingredients orsubstances, which are generally regarded as safe for human (as well asother mammals) consumption. Non-limiting examples of materials, whichcan serve as physiologically-acceptable carriers ornutritionally-acceptable or pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; (21) other non-toxic compatible substancesemployed in pharmaceutical formulations, and the like. Further, theterms ‘nutritionally-acceptable’ and ‘pharmaceutically acceptable’ asused herein refer to those compositions or combinations of agents,materials, or compositions, and/or their dosage forms, which are withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The term “homeostasis” refers to the property of a system in whichvariables are regulated so that internal conditions remain stable andrelatively constant. All animals regulate their blood glucoseconcentration. Glucose regulation in the body is a process of keepingthe body in “glucose homeostasis”. Mammals regulate their blood glucosewith different hormones (e.g., insulin, glucagon, Glucagon like peptide1, catecholamine and many others), and different nervous routes (e.g,nervous relay, gut to brain to peripheral organ axis). The human bodymaintains glucose levels constant most of the day, even after a 24-hourfast. Even during long periods of fasting, glucose levels are reducedonly very slightly. Insulin, secreted by the beta cells of the pancreas,effectively transports glucose to the body's cells by instructing thosecells to keep more of the glucose for their own use. If the glucoseinside the cells is high, the cells will convert it to the insolubleglycogen to prevent the soluble glucose from interfering with cellularmetabolism. Ultimately this lowers blood glucose levels, and insulinhelps to prevent hyperglycemia. When insulin is deficient or cellsbecome resistant to it, diabetes occurs. Glucagon, secreted by the alphacells of the pancreas, encourages cells to break down stored glycogen orconvert non-carbohydrate carbon sources to glucose via gluconeogenesis,thus preventing hypoglycemia. Numerous other factors and hormones areinvolved in the control of glucose metabolism (e.g., Glucagon likepeptide 1, catecholamine and many others). Different mechanismsinvolving nervous routes are also contributing to this complexregulation.

“Cholesterol homeostasis” is a mechanism that contributes to the processof maintaining a balanced internal state of cholesterol within a livingorganism. Cholesterol, an essential biological molecule in the humanbody system, performs various physiological functions such as acting asa precursor for the production of bile acids, vitamin D, and steroidhormones. It also functions as a critical structural element in the cellmembrane of every cell present in the body. Despite cholesterol'sbeneficial and necessary functions, an upset in cholesterol homeostasiscan cause an increased risk of heart disease as well as upsetting otherhomeostatic feedback systems associated with cholesterol metabolism. Themost conspicuous organ that controls cholesterol homeostasis is theliver because it not only biosynthesizes cholesterol released into thecirculatory system, but breaks down potentially harmful, free-floatingcholesterol from the bloodstream. HDLs are beneficial in maintainingcholesterol homeostasis because they pick up and deliver potentiallydangerous cholesterol directly back to the liver where it is synthesizedinto harmless bile acids used by the digestive system. LDLs operate lessbeneficially because they tend to deposit their cholesterol in bodycells and on arterial walls. It is excessive levels of LDLs that havebeen shown to increase risk for cardiovascular disease. In healthysubjects, cholesterol homeostasis is tightly regulated by complexfeedback loops. In this case, if the healthy subject eats copiousamounts of dietary cholesterol, biosynthesis in the liver is greatlyreduced to keep balance. In a healthy subject who has a high baselineLDL level, either from years of poor diet habits or other genetic ormedical conditions, the feedback loop and systemic coping mechanism maybe overwhelmed by the same copious intake, causing dangerous homeostaticimbalance.

“Triglyceride homeostasis” is a mechanism that contributes to theprocess of maintaining a balanced internal state of triglycerides withina living organism. Triglyceride metabolism is of great clinicalrelevance. Hypertriglyceridemia denotes high (hyper-) blood or serumlevels (-emia) of triglycerides, the most abundant fatty molecules.Elevated levels of triglycerides are associated with atherosclerosis,even in the absence of hypercholesterolemia (high cholesterol levels),and predispose to cardiovascular disease. High triglyceride levels alsoincrease the risk of acute pancreatitis. Additionally, elevations andincreases in TG levels over time enhance the risk of developingdiabetes. It has been shown that insulin resistance is associated withhigh levels of triglycerides (TGs).

The term ‘about’, as used herein indicates a range of normal tolerancein the art, for example within 2 standard deviations of the mean. Theterm ‘about’ can be understood as encompassing values that deviate atmost 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or0.01% of the indicated value.

The terms ‘comprising’ or ‘to comprise’ and their conjugations, as usedherein, refer to a situation wherein said terms are used in theirnon-limiting sense to mean that items following the word are included,but items not specifically mentioned are not excluded. It alsoencompasses the more limiting verb ‘to consist essentially of’ and ‘toconsist of’.

Reference to an element by the indefinite article ‘a’ or ‘an’ does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there be one and only one ofthe elements. The indefinite article ‘a’ or ‘an’ thus usually means ‘atleast one’.

DETAILED DESCRIPTION

The inventors have found that the polypeptide Amuc-1100, or variantsthereof as taught herein, are capable of modulating and/or promoting thegut immune system function and/or maintaining and/or restoring and/orincreasing the physical integrity of the gut mucosal barrier, and/or ofmaintaining and/or restoring and/or improving glucose and/or cholesteroland/or triglyceride homeostasis in a mammal (e.g. human). The inventorsalso found that this polypeptide is present outside the cells encoded byAkkermansia muciniphila supporting its role in signalling.

Without wishing to be bound by any theories, it is believed that suchbeneficial effects result from the ability of the polypeptides of theinvention to interact with the TLR2 signalling pathway present at thesurface of immune cells located in the vicinity of the gut mucosalbarrier of a mammal. More specifically, the present inventors found thatthe polypeptides as taught herein are capable of interacting with to theTLR2 present at the surface of an immune cell and/or modulating and/orstimulating the TLR2-signaling pathway in an immune cell located in thevicinity of the gut mucosal barrier, so as to stimulate the secretion ofcytokines (e.g. IL-6, IL-8, and IL-10) from said immune cells.

Further, the present inventors found that the polypeptides, includingvariants thereof, as taught herein, are capable of modulating and/orincreasing the transepithelial resistance of the gut mucosal barrier ofa mammal. Since increased transepithelial resistance measurement servesas an index of decreased permeability of the gut mucosal barrier, it isbelieved that the polypeptides, including variants thereof, as taughtherein are capable of modulating the physical integrity of the gutmucosal barrier, particularly at the level of the tight junctionsbetween epithelial cells.

Combined together, these effects are believed to result in an improvedor increased gut mucosal immune system function (e.g. greater release ofcytokines at the gut mucosal barrier) as well as improved or increasedphysical integrity of the gut mucosal barrier, particularly at the levelof the connection between gut epithelial cells (i.e. via tighter tightjunctions between cells).

Additionally, it was found that treatment of HFD-fed mice with Amuc-1100caused a prominent decrease in body weight and fat mass gain withoutaffecting food intake. Treatment with Amuc-1100 also corrected theHFD-induced hypercholesterolemia, with a significant decrease in serumHDL-cholesterol and a similar trend for LDL-cholesterol. Further,administration of Amuc-1100 reduced glucose intolerance with the samepotency as the live Akkermansia muciniphila bacterium.

Finally, it is known that metformin stimulates the growth of Akkermansia(Lee H and Ko G, Appl Environ Microbiol. 2014 October; 80(19):5935-43)and hence it is likely that Akkermansia and its extracellular peptidessuch as Amuc-1100 may have a similar effect as metformin on gestationaldiabetes and on preeclampsia (Syngelaki et al. N Engl J Med. 2016 Feb.4; 374(5):434-43).

Polypeptides

The present disclosure teaches an isolated polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or an amino acid sequence comprisingat least 50% amino acid sequence identity to the amino acid sequence ofSEQ ID NO:1 over the entire length, said polypeptide being capable ofeffecting immune signalling and/or affecting intestinal barrier functionand/or affecting glucose and/or cholesterol homeostasis. The polypeptidetaught herein may be capable of binding to the toll like receptor 2(TLR2).

In one embodiment, the polypeptides and variants thereof as taughtherein are capable of stimulating the TLR2 signalling pathway in a cell,stimulating the release of cytokines from a cell (e.g. IL-6, IL-8, IL-10and the like) and/or increasing transepithelial resistance (TER) ofmammalian, e.g., human, cells, and/or improving the metabolic or immunestatus of a mammal, e.g., mouse or human.

The polypeptide taught herein may also be referred to as ‘Amuc-1100protein’ or ‘Amuc-1100 polypeptide’. It is to be understood that theterm ‘Amuc-1100 protein’ or ‘Amuc-1100 polypeptide’ or ‘polypeptide astaught herein’ also includes variants of the Amuc-1100 protein havingthe amino acid sequence of SEQ ID NO:1, the amino acid sequences of saidvariants having more than 50%, preferably more than 55%, more than 60%,more than 65%, more than 70%, preferably more than 75%, more than 80%,more than 85%, more than 90%, more than 95%, preferably more than 96%,preferably more than 97%, preferably more than 98%, and preferably morethan 99% sequence identity with the amino acid sequence of SEQ ID NO:1.Variants of the Amuc-1100 polypeptide having the amino acid sequence ofSEQ ID NO:1 also include polypeptides, which have been derived, by wayof one or more amino acid substitutions, deletions or insertions, fromthe polypeptide having the amino acid sequence of SEQ ID NO:1.Preferably, such polypeptides comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20,15 amino acid substitutions, deletions or insertions as compared to thepolypeptide having the amino acid sequence of SEQ ID NO:1.

The polypeptide as taught herein may be preceded by a N terminal signalsequence stimulating secretion of the polypeptide from the cell. In anembodiment, the N terminal signal sequence may be a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, which is thepredicted naturally occurring N terminal signal sequence of theAmuc-1100 polypeptide. However, other N terminal signal sequencescapable of allowing Amuc-1100 to be secreted from a cell may also beemployed. For example, a truncated version or expanded version of thepredicted naturally occurring N terminal signal sequence of theAmuc-1100 polypeptide may be employed, as long as such N terminal signalsequence is capable of allowing Amuc-1100 to be secreted from a cell.Alternatively, a non-naturally occurring N terminal signal sequence maybe employed. The skilled person is capable of identifying N terminalsignal sequences that are suitable for use in the present invention.Thus, a polypeptide of the present invention may comprise the amino acidsequence of SEQ ID NO:3 N terminal from its amino acid sequence.

Amino acid sequence identity may be determined by any suitable meansavailable in the art. For instance, amino acid sequence identity may bedetermined by pairwise alignment using the Needleman and Wunschalgorithm and GAP default parameters as defined above. It is alsounderstood that many methods can be used to identify, synthesize orisolate variants of the polypeptides as taught herein, such as westernblot, immunohistochemistry, ELISA, amino acid synthesis, and the like.

It is also understood that any variants of the Amuc-1100 polypeptides astaught herein exert the same function and/or have the same activity asthe polypeptide Amuc-1100 as taught herein. The functionality oractivity of any Amuc-1100 polypeptides or variants thereof may bedetermined by any known methods in the art, which the skilled personwould consider suitable for these purposes.

Polynucleotides

The present disclosure also teaches a nucleic acid molecule, such as anisolated, synthetic or recombinant nucleic acid molecule, comprising anucleic acid sequence selected from the group of:

-   (a) a nucleic acid sequence having at least 50% sequence identity    with SEQ ID NO: 2 over the entire length; and-   (b) a nucleic acid sequence that encodes the polypeptide as taught    herein.

The term “isolated nucleic acid molecule” (e.g. cDNA, genomic DNA orRNA) includes naturally occurring, artificial or synthetic nucleic acidmolecules. The nucleic acid molecules may encode any of the polypeptidesas taught herein. Said nucleic acid molecule may be used to produce thepolypeptides as taught herein. Due to the degeneracy of the genetic codevarious nucleic acid molecules may encode the same polypeptide (e.g. apolypeptide comprising the amino acid sequence of SEQ ID NO:1).

In an embodiment, the isolated nucleic acid molecules as taught hereininclude any variant nucleic acid molecules, which encompass any nucleicacid molecules comprising a nucleotide sequence having more than 50%,preferably more than 55%, preferably more than 60%, preferably more than65%, preferably more than 70%, preferably more than 75%, preferably morethan 80%, preferably more than 85%, preferably more than 90%, preferablymore than 95%, preferably more than 96%, preferably more than 97%,preferably more than 98%, and preferably more than 99% sequence identitywith the nucleic acid sequence of SEQ ID NO:2. Variants also includenucleic acid molecules, which have been derived, by way of one or morenucleic acid substitutions, deletions or insertions, from the nucleicacid molecule having the nucleic acid sequence of SEQ ID NO:2.Preferably, such nucleic acid molecules comprise from 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30,25, 20, 15 nucleic acid substitutions, deletions or insertions ascompared to SEQ ID NO:2.

Sequence identity may be determined by any suitable means available inthe art. For instance, bioinformatics may be used to perform pairwisealignment between nucleic sequences to identify regions of similaritythat may be due to functional, structural, or evolutionary relationshipsbetween the sequences. It is also understood that many methods can beused to identify, synthesize or isolate variants of the polynucleotideas taught herein, such as nucleic acid hybridization, PCR technology, insilico analysis and nucleic acid synthesis, and the like.

It is further understood that any nucleic acid molecule as taught hereinmay encode a polypeptide as taught herein.

In an embodiment, the nucleic acid molecule is a nucleic acid moleculehaving the nucleic acid sequence as set forth in SEQ ID NO:2.

Alternatively, the isolated nucleic acid molecule may be a nucleic acidmolecule that hybridizes under stringent conditions with the nucleicacid molecules as taught herein and encoding a polypeptide as taughtherein. For instance, such nucleic acid sequence may be advantageouslyused in screening assays aimed to detect the presence or absence of anyhomologs of the nucleic acid molecules of the invention in a cell or inan organism or to detect a decreases or increase in the expression ofthe nucleic acid molecules as taught herein, in a cell or in anorganism.

The nucleic acid molecule as taught herein may encompass a nucleic acidmolecule encoding a N terminal signal sequence that is suitable forstimulating secretion of the polypeptide as taught herein from its hostcell. Said N terminal signal sequence encoding nucleic acid molecule maycomprise the nucleic acid sequence as set forth in SEQ ID NO:4.

In an embodiment, the nucleic acid molecule as taught herein may becomprised in a chimeric gene, wherein said nucleic acid molecule isoperably linked to a promoter. Thus the present inventions also relatesto a chimeric gene comprising the nucleic acid molecule as taughtherein.

Any promoters known in the art, and which are suitable for linkage withthe nucleic acid molecules as taught herein may be used. Non-limitingexamples of suitable promoters include promoters allowing constitutiveor regulated expression, weak and strong expression, and the like. Anyknown methods in the art may be used to include the nucleic acidmolecule as taught herein in a chimeric gene.

It may be advantageous to operably link the nucleic acid molecule astaught herein to a so-called ‘constitutive promoter’.

Alternatively, it may be advantageous to operably link thepolynucleotides and variants thereof as taught herein to a so-called‘inducible promoter’. An inducible promoter may be a promoter that isphysiologically (e.g. by external application of certain compounds)regulated.

The chimeric gene as taught herein may be comprised in a ‘vector’ or‘nucleic acid construct’. Thus the present invention also related tovectors comprising the chimeric gene as taught herein or the nucleicacid molecule as taught herein.

In an aspect, the present invention relates to a host cell that has beengenetically modified to comprise, e.g., in its genome, a nucleic acidmolecule as taught herein, a chimeric gene as taught herein or a vectorsas taught herein.

The genetically modified host cell as taught herein may be used toproduce in vitro, ex vivo and/or in vitro, the polypeptides and variantsthereof as taught herein within the host cell cytoplasm or released fromthe cells by any means. The polypeptides as taught herein may, inparticular, be expressed as a soluble or secreted molecule. Thegenetically modified host cells as taught herein can be any host cellssuitable for transformation procedures or genetic engineeringprocedures. Non-limiting examples of suitable host cells includecultivable cells, such as any prokaryotic or eukaryotic cells. In anembodiment, the AMUC-1100 polypeptide is expressed in bacteria, such asEscherichia coli.

In an embodiment, the host cell as taught herein may be any cell thatnaturally expresses the polypeptide or variant thereof taught herein. Insuch case, the host cell may overexpress the polypeptide or variantthereof as taught herein.

In yet an embodiment, the host cell as taught herein may be any cellthat does not naturally express the polypeptide or variants thereof astaught herein.

In an embodiment, the host cell as taught herein does not belong to thespecies Akkermansia muciniphila.

In another embodiment, the host cell may belong to the speciesAkkermansia muciniphila, and is genetically modified to compriseadditional copies of the nucleic acid molecules taught herein, or tocomprise a chimeric gene or vector as taught herein. Such Akkermansiamuciniphila cell may overexpress the Amuc-1100 polypeptide or a variantthereof taught herein.

The host cell as taught herein may be genetically modified using anyknown methods in the art. For instance, the host cells or organisms astaught herein may be genetically modified by a method comprising thestep of

a) transforming the host cell with a nucleic acid molecule as taughtherein, such as an isolated, synthetic or recombinant nucleic acidmolecule comprising a nucleic acid sequence selected from the group of anucleic acid sequence having at least 50% sequence identity with SEQ IDNO: 2 over the entire length; and a nucleic acid sequence capable ofencoding the polypeptides and variants thereof as taught herein.b) culturing said host cell under conditions suitable to allowexpression of the nucleic acid molecule as taught herein and/orproduction of the polypeptide or a variant thereof as taught herein;c) optionally, screening for host cells capable of expressing thenucleic acid molecule as taught herein and/or producing the polypeptideor a variant thereof as taught herein.

In a preferred embodiment, the host cells or organisms as taught hereinmay be transformed with a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:2, or a variant thereof as taught herein.

In an embodiment, the genetically modified host cell as taught hereinmay belong to a species of bacteria that naturally occurs or lives inthe vicinity of or within the gut mucosal barrier of a mammal. Saidspecies of bacteria are often referred to as ‘gut mucosal-associatedbacteria species’. Non-limiting examples of ‘gut mucosal-associatedbacteria species’ include Akkermansia muciniphila (ATTC BAA-835),Faecalibacterium prausnitzii (A2-165), Lactobacillus rhamnosus (ATCC53103) and Bifidobacterium breve (DSM-20213).

In certain embodiments, it may be advantageous to genetically modify agut mucosal-associated bacteria with any of the polynucleotides andvariants thereof as taught herein, for instance to express oroverexpress the polynucleotides as taught herein or to produce oroverproduce the polypeptides as taught herein, directly into thevicinity of, or within the gut mucosal barrier of a mammal (e.g. human).In a preferred embodiment, the gut mucosal-associated bacteria may byany bacteria from the species Akkermansia muciniphilla. Suchoverproduction may be realized by genetic modification tools involvingrecombinant DNA technologies, genome editing such as by using toolsbased on CRISPR/cas-like systems, or by classical mutation selectionsystems.

In an embodiment, the genetically modified host cell may be anybacteria, particularly one which is not from a species of bacteria thatnaturally occurs or lives in the vicinity of or within the gut mucosalbarrier of a mammal. Non-limiting examples of such bacteria include anybeneficial isolated intestinal bacterial strains, e.g. probioticbacteria, particularly strains selected from the genera Lactococcus,Lactobacillus, or Bifidobacterium may be used. In addition, strictanaerobic intestinal bacteria may be used such as those belonging to thegenera known to occur in the human intestinal tract (Rajilic-Stojanovic& de Vos, The first 1000 cultured species of the human gastrointestinalmicrobiota. FEMS Microbiol Rev. 38: 996-1047).

Methods for Producing the Polypeptide

In a further aspect, the present invention relates to a method forproducing the polypeptides, including variants, as taught herein,comprising the steps of:

(a) culturing a host cell as taught herein under conditions permittingproduction of the polypeptide or a variant thereof as taught herein; and(b) optionally, isolating the polypeptide produced in step (a).

In step (a), the host cell as taught herein may be cultured according toany known culturing methods and on any known culture medium. The skilledperson will be able to select a suitable host cell and will be able toestablish suitable conditions allowing production of the polypeptide.

Alternatively, the polypeptide may be produced by a method comprisingthe steps of:

(a) culturing bacteria of the species Akkermansia muciniphila in asuitable culture medium; and(b) optionally, isolating the polypeptide produced in step (a).

The polypeptide produced in steps (a) of the methods above may beisolated by any known methods in the art. The skilled person will becapable of isolating the polypeptide produced from such culture medium.

Suitable culture media are, for example, taught by Derrien et al. (2004,Int. J. Syst. Evol. Microbiol. 54: 1469-76). Derrien et al. teach thatA. muciniphila strain Muc^(T) was isolated and grown on a basalanaerobic medium containing hog gastric mucin as the sole carbon andnitrogen source. The authors also teach that A. muciniphila can be grownon rich media, such as Columbia Broth (CB) and Brain Heart Infusion(BHI) broth or basal medium with glucose and high concentrations ofcasitone and yeast-extract. Similarly, Lukovac et al. (mBio teaches thegrowth of A. muciniphila in a basal medium containing glucose andfucose, as well as high amounts of casitone (2014, mBio 01438-14)

Methods for Screening Bacteria

In further aspect, the present invention relates to a method fordetecting the presence or absence, in a bacteria, of a polynucleotide astaught herein, comprising the step of:

(a) providing a nucleic acid molecule that is capable of hybridizingunder stringent conditions to a nucleic acid molecule having the nucleicacid sequence of SEQ ID NO:2 or having a nucleic acid sequencecomprising at least 50% sequence identity to the nucleic acid sequenceof SEQ ID NO:2 over the entire length;(b) detecting the nucleic acid molecule of step (a) to identify abacteria comprising the nucleic acid molecule having the nucleic acidsequence of SEQ ID NO:2 or a nucleic acid sequence comprising at least50% nucleic acid sequence identity to the nucleic acid sequence of SEQID NO:2 over the entire length.

The present disclosure also relates to a method for detecting thepresence or absence, in a bacteria, of a polypeptide or variants thereofas taught herein, comprising the step of:

-   -   (a) providing an antibody that is capable of binding to a        polypeptide comprising the amino acid sequence of SEQ ID NO:1 or        an amino acid sequence comprising at least 50% sequence identity        to the amino acid sequence of SEQ ID NO:1;    -   (b) detecting the antibody of step (a) to identify bacteria        comprising a polypeptide having the amino acid sequence of SEQ        ID NO:1 or an amino acid sequence comprising at least 50%        sequence identity to the amino acid sequence of SEQ ID NO:1.

In an embodiment, the nucleic acid of step (a) and/or the antibody ofstep (c) are labelled (e.g. fluorescent, radioactive labels, etc.) tofacilitate detection.

Compositions

In a further aspect, the present inventions relates to a compositioncomprising any of the polypeptides as taught herein. In a preferredembodiment, the polypeptide has the amino acid sequence of SEQ ID NO: 1.

In a yet further aspect, the present invention relates to a compositioncomprising a host cell as taught herein. The host cell may be present inan amount ranging from about 10⁴ to about 10¹⁵ colony forming units(CFU). For instance, an effective amount of the host cell may be anamount of about 10⁵ CFU to about 10¹⁴ CFU, preferably about 10⁶ CFU toabout 10¹³ CFU, preferably about 10⁷ CFU to about 10¹² CFU, morepreferably about 10⁸ CFU to about 10¹² CFU. The host cell may be viableor may be dead. The effectiveness of the host cell correlates with thepresence of the polypeptide as taught herein.

In an embodiment, the composition as taught herein further comprises acarrier, e.g., a physiologically acceptable carrier or apharmaceutically acceptable carrier or an alimentarily acceptablecarrier or a nutritionally acceptable carrier. The carrier may be anyinert carrier. For instance, non-limiting examples of suitablephysiologically or pharmaceutically acceptable carriers include any ofwell-known physiological or pharmaceutical carriers, buffers, diluents,and excipients. It will be appreciated that the choice for a suitablephysiological or pharmaceutical carrier or alimentary carrier ornutritional carrier will depend upon the intended mode of administrationof the composition as taught herein (e.g., oral) and the intended formof the composition (e.g. beverage, yogurt, powder, capsules, and thelike). The skilled person knows how to select a suitable carrier, e.g.,physiologically acceptable carrier or a nutritionally acceptable carrieror a pharmaceutically acceptable carrier, which is suitable for orcompatible with the compositions as taught herein.

In an embodiment, the compositions as taught herein may be anutritional, or alimentary, composition. For instance, the compositionas taught herein may be a food, food supplement, feed, or a feedsupplement such as a dairy product, e.g., a fermented dairy product,such as a yogurt or a yogurt drink. In this case, the composition maycomprise a nutritionally acceptable or alimentarily acceptable carrier,which may be a suitable food base.

In an embodiment, the compositions as taught herein may be apharmaceutical composition. The pharmaceutical composition may also befor use as a supplement (e.g. food supplement). The pharmaceuticalcomposition as taught herein may comprise a pharmaceutical,nutritionally or alimentarily or physiologically-acceptable carrier, inaddition to the polypeptide as taught herein and/or host cells as taughtherein. The preferred form will depend on the intended mode ofadministration and (therapeutic) application. The carrier may be anycompatible, physiologically-acceptable, non-toxic substances suitable todeliver the polypeptide as taught herein and/or host cell as taughtherein to the GI tract of a mammal (e.g. human), preferably in thevicinity of or within the gut mucosal barrier (more preferably the colonmucosal barrier) in a mammal. For example, sterile water, or inertsolids may be used as a carrier, usually complemented with apharmaceutically acceptable adjuvant, buffering agent, dispersing agent,and the like.

The composition as taught herein may be in liquid form, e.g. astabilized suspension of the polypeptide as taught herein or host cellas taught herein, or in solid form, e.g., a powder of lyophilized hostcells as taught herein. In case the host cells as taught herein arelyophilized, a cryoprotectant such as lactose, trehalose or glycogen maybe employed. For oral administration, polypeptides as taught herein orlyophilized host cells as taught herein may be administered in soliddosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The polypeptideas taught herein or host cell as taught herein may be encapsulated incapsules such as gelatin capsules, together with inactive ingredientsand powder carriers, such as e.g. glucose, lactose, sucrose, mannitol,starch, cellulose or cellulose derivatives, magnesium stearate, stearicacid, sodium saccharin, talcum, magnesium carbonate and the like.

In an embodiment, the compositions as taught herein may comprise one ormore ingredients, which are suitable for promoting survival and/orviability and/or maintaining the and/or integrity of the polypeptide astaught herein and/or the host cell as taught herein during storageand/or during exposure to bile and/or during passage through the GItract of a mammal (e.g. a human). Non-limiting examples of suchingredients include an enteric coating, and controlled release agentsallowing passage through the stomach. The skilled person knows how toselect suitable ingredients for ensuring that the active component (beit a polypeptide or a host cell) receives its intended destination,where it exerts its action.

In an embodiment, the compositions as taught herein may further comprisea mucosal binding agent or mucosal binding polypeptide. The term‘mucosal binding agent’ or ‘mucosal binding polypeptide’ as used hereinrefers to an agent or a polypeptide that is capable of attaching itselfto the gut mucosal surfaces of the gut mucosal barrier of a mammal (e.g.human).

Alternatively, use can be made of specific docking systems to attach theAmuc-1100 polypeptide to Amuc-1100 producing cells or even nonAmuc-1100-producing cells that are either alive or dead. The binding canbe either at the C- or N-terminus, whatever seems to be most efficient,while also the use of spacer peptides has been described. Examplesinclude the use of LysM-based peptidoglycan binding systems (VisweswaranG R et al. 2014, Appl Microbiol Biotechnol. 98:4331-45). Moreover, avariety of mucosal binding polypeptides have been disclosed in the art.Non-limiting examples of mucosal binding polypeptide include bacterialtoxin membrane binding subunits including such as the B subunit ofcholera toxin, the B subunit of the E. coli heat-labile enterotoxin,Bordetella pertussis toxin subunits S2, S3, S4 and/or S5, the B fragmentof Diphtheria toxin and the membrane binding subunits of Shiga toxin orShiga-like toxins. Other suitable mucosal binding polypeptides includebacterial fimbriae proteins such as including E. coli fimbria K88, K99,987P, F41, FAIL, CFAIII ICES1, CS2 and/or CS3, CFAIIV ICS4, CS5 and/orCS6), P fimbraiae, or the like. Other non-limiting examples of fimbriaeinclude Bordetella pertussis filamentous hemagglutinin, Vibrio choleraetoxin-coregulate pilus (TCP), Mannose-sensitive hemagglutinin (MSHA),fucose-sensitive hemagglutinin (PSHA), and the like. Still othermucosal-binding agents include viral attachment proteins includinginfluenza and sendai virus hemagglutinins and animal lectins orlectin-like molecules including immunoglobulin molecules or fragmentsthereof, calcium-dependant (C-type) lectins, selectins, collectins orhelix pomatis hemagglutinin, plant lectins with mucosa-binding subunitsinclude concanavalin A, wheat-germ agglutinin, phytohemagglutinin,abrin, ricin and the like. The advantage of this delivery is that oneobviates the use of a living recombinant organism.

Although not essential, it may be advantageous to add one or moremucosal binding agent or mucosal binding polypeptide to the compositionas taught herein so as to target the polypeptide as taught herein or thehost cell as taught herein to the gut mucosal barrier.

The compositions as taught herein may further comprise ingredientsselected from the group consisting of prebiotics, probiotics,carbohydrates, polypeptides, lipids, vitamins, minerals, medicinalagents, preservative agents, antibiotics, or any combination thereof.

In one embodiment, the composition as taught herein may further compriseone or more ingredients, which further enhance the nutritional valueand/or the therapeutic value the compositions as taught herein. Forinstance, it may be advantageous to add one or more ingredients (e.g.nutritional ingredients, veterinary or medicinal agents etc.) selectedfrom proteins, amino acids, enzymes, mineral salts, vitamins (e.g.thiamine HCl, riboflavin, pyridoxine HCl, niacin, inositol, cholinechloride, calcium pantothenate, biotin, folic acid, ascorbic acid,vitamin B12, p-aminobenzoic acid, vitamin A acetate, vitamin K, vitaminD, vitamin E, and the like), sugars and complex carbohydrates (e.g.water-soluble and water-insoluble monosaccharides, disaccharides, andpolysaccharides), medicinal compounds (e.g. antibiotics), antioxidants,trace element ingredients (e.g. compounds of cobalt, copper, manganese,iron, zinc, tin, nickel, chromium, molybdenum, iodine, chlorine,silicon, vanadium, selenium, calcium, magnesium, sodium and potassiumand the like). The skilled person is familiar with methods andingredients that are suitable to enhance the nutritional and/ortherapeutic/medicinal value of the compositions as taught herein.

In an embodiment, the host cell may be incorporated in lyophilized form,or microencapsulated form (reviewed by, for example, Solanki et al.BioMed Res. Int. 2013, Article ID 620719), or any other form preservingthe activity and/or viability of the host cell (e.g. bacterial strain).

Methods of Treatment

In another aspect, the present invention relates to methods for treatingand/or preventing a disorder or condition selected from the group ofobesity, metabolic syndrome, insulin-deficiency or insulin-resistancerelated disorders, type 2 diabetes, type 1 diabetes, gestationaldiabetes, preeclampsia, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism,atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholicfatty liver disease, alcoholic fatty liver disease, hyperglycemia,hepatic steatosis, dyslipidaemias, dysfunction of the immune systemassociated with obesity (weight gain), allergy, asthma, autism,parkinson's disease, multiple sclerosis, neurodegenerative diseases,depression, other diseases related to compromised barrier function,wound healing, behavioural disorders, alcohol dependence, cardiovasculardiseases, high cholesterol, elevated triglycerides, atherosclerosis,sleep apnoea, osteoarthritis, gallbladder disease, cancer, andconditions altering the physical integrity of the gut mucosal barriersuch as food allergies, immaturity of the gut, e.g., due to a baby beingborn prematurely, exposure to radiation, chemotherapy and/or toxins,autoimmune disorders, malnutrition, sepsis, and the like, in a mammal;methods for promoting weight loss in a mammal; methods for promotinganti-inflammatory activity in the gut of a mammal; methods for promotinggut mucosal immune system function in a mammal; methods for maintaining,restoring and/or improving glucose and/or cholesterol and/ortriglyceride homeostasis; and methods for maintaining, restoring and/orincreasing the physical integrity of the mucosal gut barrier in amammal. The methods comprise the step of administering to a mammal inneed thereof, an effective amount of a polypeptide as taught herein, ahost cell as taught herein or a composition as taught herein.

In one embodiment, the polypeptide as taught herein, a host cell astaught herein or a composition as taught herein may be administered byany known methods of administration. For instance, the compositions astaught herein may be administered orally, intravenously, topically,enterally or parenterally. It is understood that the modes or routes ofadministration will depend on the case at hand (e.g. age of the subject,desired location of the effects, disease conditions and the like) aswell as on the intended form of the composition (e.g. pill, liquid,powder etc.).

In a preferred embodiment, the polypeptide as taught herein, a host cellas taught herein or a composition as taught herein are administeredorally.

Uses

In a further aspect, the present invention relates to the use of thenucleic acid molecule as taught herein, chimeric gene as taught hereinand/or vectors as taught herein for producing the polypeptides as taughtherein and/or for generating the host cells as taught herein. Thepolypeptide as taught herein and/or the host cell as taught herein mayhave enhanced ability to interact with the TLR2 receptor on a celland/or may have an enhanced ability to stimulate TLR2 signalling pathwayin a cell, and/or may have an enhanced ability to stimulate productionof cytokines, particularly IL-1β, IL-6, IL-8, IL-10 and TNF-α, from acell, and/or may have an enhanced ability to increase TER of mammalian,e.g., human, cells, as compared to a host cell (e.g. bacteria) notgenetically modified with the polynucleotides, chimeric genes or vectorsas taught herein.

In a further aspect, the present invention relates to the polypeptide astaught herein, host cells as taught herein or composition as taughtherein for use as a medicament; particularly for use in promoting gutmucosal immune system function or for maintaining, restoring and/orincreasing the physical integrity of the gut mucosal barrier in amammal; for maintaining, restoring and/or improving glucose and/orcholesterol and/or triglyceride homeostasis in a mammal; for use inpreventing and/or treating a disorder or condition selected from thegroup consisting of obesity, such as diet-induced obesity, metabolicsyndrome, insulin-deficiency or insulin-resistance related disorders,type 2 diabetes, type 1 diabetes, gestational diabetes, preeclampsia,inflammatory bowel disease (IBD), irritable bowel syndrome (IBS),glucose intolerance, abnormal lipid metabolism, atherosclerosis,hypertension, cardiac pathology, stroke, non-alcoholic fatty liverdisease, alcoholic fatty liver disease, hyperglycemia, hepaticsteatosis, dyslipidaemias, dysfunction of the immune system associatedwith obesity (weight gain), allergy, asthma, autism, parkinson'sdisease, multiple sclerosis, neurodegenerative diseases, depression,other diseases related to compromised barrier function, wound healing,behavioural disorders, alcohol dependence, cardiovascular diseases, highcholesterol, elevated triglycerides, atherosclerosis, sleep apnoea,osteoarthritis, gallbladder disease, cancer, and conditions altering thephysical integrity of the gut mucosal barrier such as food allergies,immaturity of the gut, e.g., due to a baby being born prematurely,exposure to radiation, chemotherapy and/or toxins, autoimmune disorders,malnutrition, sepsis, and the like, in a mammal; for use in promotinganti-inflammatory activity in the gut of a mammal; or for use inpromoting weight loss in a mammal.

In an embodiment, the mammal, e.g., human, may be of any age group (e.g.infants, adults, elderly) and of any gender (male and female). In anembodiment, the mammal may be an infant (e.g. new-borns, babies,toddlers etc.), particularly an infant, which was born prematurely.

The mammal may be any mammal, for example, humans, non-human primates,rodents, cats, dogs, cow, horses, and the like. In a preferredembodiment, the mammal is a human being.

The present invention is further illustrated, but not limited, by thefollowing examples. From the above discussion and these examples, oneskilled in the art can ascertain the essential characteristics of thepresent invention, and without departing from the teaching and scopethereof, can make various changes and modifications of the invention toadapt it to various usages and conditions. Thus, various modificationsof the invention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

DESCRIPTION OF THE FIGURE

FIG. 1 shows: A) Total body weight gain (g) (n=8-10). B) Total fat massgain (g) measured by Time domain-Nuclear magnetic resonance (n=8-10). C)daily food intake. D) Plasma VLDL, LDL and HDL cholesterol levels(n=8-10). E) Plasma glucose (mg dl⁻¹) profile and F) mean area under thecurve (AUC) measured between −30 and 120 min after glucose loading(mg.dr¹.min⁻¹; n=8-10). G) Ratio of the control and insulin-stimulatedp-IRβ on the loading control as measured by densitometry (n=3-5). H andI) Ratio of the control and insulin-stimulated p-Akt^(thr308) andp-Akt^(ser473) on the loading control as measured by densitometry(n=3-5).

SEQUENCE LISTING

SEQ ID NO:1: Amino acid sequence of the Amuc-1100 polypeptideSEQ ID NO: 2: Nucleotide sequence encoding the Amuc-1100 polypeptideSEQ ID NO:3: Amino acid sequence of the predicted N-terminal signalsequence of Amuc-1100 polypeptideSEQ ID NO:4: Nucleotide sequence of the predicted N-terminal signalsequence of Amuc-1100 polypeptide

Examples Example 1: Generation of Bacteria Genetically Modified toProduce Amuc-1100 Proteins Method:

The polynucleotide encoding the mature Amuc-1100 (nucleotide sequence ofSEQ ID NO:2) was cloned into E. coli TOP10 with a C-terminal His-Tagunder control of the inducible T7 promoter of pET28-derivatives andintroduced into E. coli BL23(DE3) for overproduction. For this purposean ATG start codon was added to the nucleotide sequence of SEQ ID NO; 2,so that the resulting polypeptide started with the amino acid sequenceMIVNS. All constructs were confirmed by Sanger sequence analysis. Theconstructs carrying the overexpressed Amuc-1100 resulted inoverproduction of soluble Amuc-1100 proteins that were purified toapparent homogeneity by Ni-column affinity chromatography and used in aconcentration of 100-300 ug/ml. The purified Amuc-1100 was used togenerate antibodies in rabbits essentially as described previously(Reunanen J et al. 2012, Appl Environ Microbiol 78:2337-44).

Results:

The results show that E. coli transformed with the polynucleotide of theinvention (SEQ ID NO:2) was able to produce the Amuc-1100 protein in asoluble form that could be isolated easily using Ni-columnchromatography as described (Tailford L E et al. 2015, Nat Commun.6:7624).

Example 2: Interaction and Stimulation of the TLR2 Signalling PathwayMethod:

In order to test the ability of Amuc-1100 to bind the TLR2 and other TLRreceptors and subsequently stimulate the TLR2 and other TLR signallingpathways, reporter cell lines expressing TLR2 and TLR4 receptors wereprepared. The ability of Amuc-1100 to bind cell lines expressing TLR2 orTLR4 and thereafter stimulate the TLR2 and/or TLR4 signaling pathway insaid cells was tested in vitro by measuring the production of NK-kB fromthe reporter cells.

Briefly, hTLR2 and hTLR4 cell lines (Invivogen, Calif., USA) were used.Stimulation of the receptors with the corresponding ligands activatesNF-κB and AP-1, which induces the production of Secreted embryonicalkaline phosphatase (SEAP), the levels of which can be measured byspectrophotometer (Spectramax). All cell lines were grown andsubcultured up to 70-80% of confluency using as a maintenance mediumDulbecco's Modified Eagle Medium (DMEM) supplemented with 4.5 g/ID-glucose, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/mlNormocin, 2 mM L-glutamine, and 10% (v/v) of heat-inactivated FetalBovine Serum (FBS). For each cell line, an immune response experimentwas carried out by adding 20 μl of Amuc-1100 suspensions. The reportercells were incubated with Amuc-1100 for 20-24 h at 37° C. in a 5% CO2incubator. Receptor ligands Pam3CSK4 (10 ng/ml for hTLR2) and LPS-EB (50ng/ml for hTLR4) were used as positive control whereas maintenancemedium without any selective antibiotics was used as negative control.SEAP secretion was detected by measuring the OD600 at 15 min, 1 h, 2 h,and 3 h after addition of 180 μL of QUANTI-Blue (Invivogen, Calif., USA)to 20 μL of induced hTLR2 and hTLR4 supernatant. Experiments wereperformed in triplicate.

Results:

The results show that Amuc-1100 was able to interact with TLR2. Further,the results show that Amuc-1100 exerted immune-stimulatory effects onreporter cells expressing TLR2, i.e. Amuc-1100 was capable ofstimulating the release of NF-κB from reporter cells.

Example 3. Stimulation of Cytokine Release from Peripheral BloodMononuclear Cells Method:

The ability of Amuc-1100 to stimulate cytokine production or releasefrom peripheral blood mononuclear cells (PBMCs) was tested in vitro.Briefly, peripheral blood of three healthy donors was received from theSanquin Blood Bank, Nijmegen, The Netherlands. Peripheral bloodmononuclear cells (PBMCs) were separated from the blood of healthydonors using Ficoll-Paque Plus gradient centrifugation according to themanufacturer's protocol (Amersham biosciences, Uppsala, Sweden). Aftercentrifugation the mononuclear cells were collected, washed in Iscove'sModified Dulbecco's Medium (IMDM)+Glutamax (Invitrogen, Breda, TheNetherlands) and adjusted to 0.5×10⁶ cells/ml in IMDM+Glutamaxsupplemented with penicillin (100 U/ml) (Invitrogen), streptomycin (100μg/ml) (Invitrogen), and 10% heat inactivated FBS (Lonza, Basel,Switzerland). PBMCs (0.5×10⁶ cells/well) were seeded in 48-well tissueculture plates. For each donor, a negative control (medium only) wasused.

The PBMCs were stimulated with A. muciniphila cells (1:10 ratio toPBMCs) either alive or heated for 10 min at 99° C.) or Amuc-1100 for 1day and subsequently the production of cytokine IL-6, IL-8, IL-10,TNF-α, IL-113 and IL-12p70 was measured in culture supernatants usingmultiple analysis (Human inflammation CBA kit, Becton and Dickinson)according to the manufacturer's protocol on a FACS CantoII (BectonDickinson) and analysed using BD FCAP software (Becton Dickinson). Thedetection limits according to the manufacturer were as follows: 3.6pg/ml IL-8, 7.2 pg/ml IL-1β, 2.5 pg/ml IL-6, 3.3 pg/ml IL-10, 3.7 pg/mlTNF-α, 1.9 pg/ml IL-12p70.

Results

The results show that, compared to the control situation (medium only),Amuc-1100 was able to stimulate the production of cytokines, i.e.increased levels of IL-113, IL-6, IL-8, IL-10 and TNF-α were observed.The level of cytokine induced by 4.5 μg/ml Amuc-1100 was at a similarlevel as that of 5×10⁶ cells of A. muciniphila either alive or in aheat-killed form (see Table 1 below).

TABLE 1 Levels of cytokine induced by Amuc-1100 and Akkermansiamuciniphila either alive or in a heat-killed form Live Heat-killedAmuc-1100 Cytokine (pg/ml) A. muciniphila A. muciniphila (4.5 μg/ml)IL-1β 894 ± 298 392 ± 71 504 ± 227 IL-6 18029 ± 309  13477 ± 2014 12508± 2362  IL-8 60018 ± 18229 54230 ± 9030 45432 ± 12507 IL-10 823 ± 310 638 ± 118 526 ± 180 TNF-α 1920 ± 349   957 ± 568 1317 ± 885  IL-12p70<2 <2 <2

Example 4: Modulation of the Transepithelial Resistance (TER) Method:

The ability of Amuc-1100 to promote the integrity of gut epithelial celllayer was assessed by measuring the ability of Amuc-1100 to stimulate orincrease TER of Caco-2 cells in vitro. Briefly, Caco-2 cells (5×10⁴cells/insert) were seeded in Millicell cell culture inserts (3 μm poresize; Millipore) and grown for 8 days. Bacterial cells were washed oncewith RPMI 1640, and applied onto the inserts at OD600 nm of 0.25(approximately 10⁸ cells) in RPMI 1640. Purified Amuc-1100 was appliedonto the inserts at concentrations of 0.05, 0.5 and 5 μg/ml. Thetransepithelial resistance was determined with a Millicell ERS-2 TERmeter (Millipore) from cell cultures at time points 0 h, and 24 h afteraddition of Amuc-1100.

Results:

The results showed that already 0.05 μg/ml of Amuc-1100 was able tosignificantly increase TER after 24 h of co-cultivation with the Caco-2cells at a similar level of approximately 10⁸ A. muciniphila cells.

Example 5: Modulation of Diet-Induced Metabolic Dysfunction

A cohort of 10-11 week-old C57BL/6J mice (n=10 per subset) was fed acontrol diet (ND) or an HF diet (HFD; 60% fat and 20% carbohydrates(kcal/100 g) D12492i, Research Diet, New Brunswick, N.J., USA) aspreviously described by Everard et al. (2013. PNAS. Vol.110(22):9066-9071). A muciniphila Muc^(T) was grown on a syntheticmedium (containing per liter deionized water: 0.4 g KH₂PO₄, 0.669 gNa₂HPO₄.2H₂O, 0.3 g NH₄Cl, 0.3 g NaCl, 0.1 g MgCl₂.6H²O, 10 g Casitone,1 mM L-threonine, 1 ml trace mineral solution, 5 mM L-fucose and 5 mMD-glucose) as described by Lucovac et al. (2014, mBio 01438-14) andconcentrated, formulated in PBS containing 25% glycerol, and stored at−80° C. as described by Everard et al. supra. A subset of mice receivingHFD additionally received, daily and by oral gavage, 2×10⁸ cfu/0.15 mlA. muciniphila suspended in sterile anaerobic PBS (HFD Akk)—since thisincluded a 10-fold dilution of the A. muciniphila, a final concentrationof 2.5% glycerol was obtained. The ND and HFD groups were treated dailywith an oral gavage of an equivalent volume of sterile anaerobic PBScontaining 2.5% glycerol, as previously described by Everard et al.,supra. A further subset of mice receiving HFD additionally receivedAmuc-1100 peptide delivered by daily oral gavage of 3.1 μg of theprotein Amuc_1100 in an equivalent volume of sterile PBS containing 2.5%glycerol. Treatment of HFD-fed mice with Amuc-1100 caused a similar oreven more prominent decrease in body weight and fat mass gain whencompared to the live A. muciniphila bacterium (FIGS. 1 A and B), withoutaffecting food intake (FIG. 1 C). Treatment with A. muciniphila orAmuc-1100 also corrected the HFD-induced hypercholesterolemia, with asignificant decrease in serum HDL-cholesterol and a similar trend forLDL-cholesterol (FIG. 1 D).

Remarkably, treatment with Amuc-1100 led to a significant decrease ofserum triglycerides when compared to untreated HFD-fed mice. Moreover,Amuc-1100 treatment also reduced the adipocyte mean diameter from 38micrometer in HFD-fed mice to 29 micrometer, a similar diameter as foundin untreated mice (27 micrometer).

Interestingly, administration of Amuc-1100 reduced glucose intolerancewith the same potency as the live bacterium (FIG. 1 E-F).

To further investigate glucose metabolism we investigated insulinsensitivity by injecting insulin in the portal vein. We analyzedinsulin-induced phosphorylation of the insulin receptor (IR) and itsdownstream mediator Akt in the liver at the threonine (Akt^(thr)) andserine (Akt^(ser)) sites (FIG. 1 G). Administration of the HFD led to adecreased phosphorylation of all proteins when compared to mice fed acontrol chow, reaching significance in the case of Akt^(thr) (FIG. 1 H).Treatment with live A. muciniphila or Amuc-1100 counteracted theseeffects, with significantly higher levels of p-IR and p-Akt^(thr) inmice treated with Amuc-1100 (FIG. 1 G-H) and significantly higher levelsof p-Akt^(ser) in mice treated with the live bacterium (FIG. 1 I) whencompared to the untreated HFD-fed mice.

1. A composition comprising an isolated polypeptide comprising the aminoacid sequence of SEQ ID NO: 1 or an amino acid sequence comprising atleast 50%6 sequence identity to the amino acid sequence of SEQ ID NO: 1over the entire length of SE ID NO: 1, said polypeptide being capable ofeffecting immune signaling and/or affecting intestinal barrier functionand/or affecting glucose and/or cholesterol and/or triglyceridehomeostasis, and a pharmaceutically or alimentary acceptable carrier. 2.The composition according to claim 1, which is a nutritional compositionor a pharmaceutical composition.
 3. A genetically modified host cellcomprising a nucleic acid molecule selected from the group of: (a) anucleic acid molecule comprising a nucleic acid sequence having at least50% sequence identity with SEQ ID NO: 2 over the entire length; and (b)a nucleic acid molecule comprising a nucleic acid sequence that encodesa polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or anamino acid sequence comprising at least 50% sequence identity to theamino acid sequence of SEQ ID NO:1 over the entire length of SEO IDNO:1, said polypeptide being capable of effecting immune signalingand/or affecting intestinal barrier function and/or affecting glucoseand/or cholesterol and/or triglyceride homeostasis.
 4. The geneticallymodified host cell of claim 3, wherein the host cell is not of thespecies Akkermansia muciniphilla.
 5. The genetically modified host cellof claim 3, wherein the host cell is of the species Akkermansiamuciniphila.
 6. A method for producing a polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or an amino acid sequence comprisingat least 50% sequence identity to the amino acid sequence of SEQ ID NO:1over the entire length of SEQ ID NO:1, said polypeptide being capable ofeffecting immune signaling and/or affecting intestinal barrier functionand/or affecting glucose and/or cholesterol and/or triglyceridehomeostasis, comprising the steps of: (a) culturing a host cell of claim3 under conditions so that the nucleic acid molecule is expressed andthe polypeptide is produced; and (b) optionally, isolating thepolypeptide produced in step (a).
 7. (canceled)
 8. A method forpromoting gut mucosal immune system function, for maintaining, restoringor improving glucose and/or cholesterol and/or triglyceride homeostasis,or for maintaining, restoring and/or increasing the physical integrityof the gut mucosal barrier in a mammal.
 9. (canceled)
 10. A method forpromoting anti-inflammatory activity in the gut of a mammal, the methodcomprising administering to a mammal in need thereof, an effectiveamount of the composition of claim
 1. 11. A method for promoting weightloss in a mammal, the method comprising administering to a mammal inneed thereof, an effective amount of the composition of claim
 1. 12. Amethod for treating and/or preventing a disorder selected from the groupof obesity, metabolic syndrome, insulin-deficiency or insulin-resistancerelated disorders, type 2 diabetes, type 1 diabetes, gestationaldiabetes, preeclampsia, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism,atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholicfatty liver disease, alcoholic fatty liver disease, hyperglycemia,hepatic steatosis, dyslipidaemias, dysfunction of the immune systemassociated with obesity (weight gain), allergy, asthma, autism,Parkinson's disease, multiple sclerosis, neurodegenerative diseases,depression, other diseases related to compromised barrier function,wound healing, behavioral disorders, alcohol dependence, cardiovasculardiseases, high cholesterol, elevated triglycerides, atherosclerosis,sleep apnea, osteoarthritis, gallbladder disease, cancer, and conditionsaltering the physical integrity of the gut mucosal barrier such as foodallergies, immaturity of the gut, e.g., due to a baby being bornprematurely, exposure to radiation, chemotherapy and/or toxins,autoimmune disorders, malnutrition, sepsis, and the like, in a mammal;for promoting weight loss in a mammal; for promoting anti-inflammatoryactivity in the gut of a mammal; for promoting gut mucosal immune systemfunction in a mammal; for maintaining, restoring or improving glucoseand/or cholesterol and/or triglyceride homeostasis; or for maintaining,restoring and/or increasing the physical integrity of the mucosal gutbarrier of a mammal, the method comprising administering to a mammal inneed thereof, an effective amount of the composition of claim
 1. 13. Amethod for producing a polypeptide comprising the amino acid sequence ofSEQ ID NO: 1 or an amino acid sequence comprising at least 50% sequenceidentity to the amino acid sequence of SEQ ID NO:1 over the entirelength of SEO ID NO:1, said polypeptide being capable of effectingimmune signaling and/or affecting intestinal barrier function and/oraffecting glucose and/or cholesterol and/or triglyceride homeostasis,comprising the steps of: (a) culturing a host cell of the speciesAkkermansia muciniphila comprises a nucleic acid molecule comprising anucleic acid sequence that encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO: 1 or an amino acid sequence comprising atleast 50%6 sequence identity to the amino acid sequence of SEQ ID NO:1over the entire length of SEQ ID NO: 1 in a suitable culture medium sothat the nucleic acid molecule is expressed and the polypeptide isproduced; and (b) optionally, isolating the polypeptide produced in step(a).
 14. The method of claim 13, wherein the polypeptide comprises theamino acid sequence set forth in SEQ ID NO:1.
 15. The method of claim13, wherein the nucleic acid molecule comprises the nucleic acidsequence set forth in SEQ ID NO:2.
 16. The composition of claim 1,wherein the polypeptide comprises the amino acid sequence set forth inSEQ ID NO:1.
 17. The genetically modified host cell of claim 3, whereinthe polypeptide comprises the amino acid sequence set forth in SEQ IDNO:1.
 18. The genetically modified host cell of claim 3, wherein thenucleic acid molecule comprises the nucleic acid sequence set forth inSEQ ID NO:2.